Comparison of two scaffolding polypeptides for the integration of different proteins in synthetic complexes derived from the Clostridium thermocellum cellulosome

The targeted integration of different polypeptides within artificial complexes derived from the Clostridium thermocellum cellulosome was investigated. Two scaffolding polypeptides, each containing two cohesin domains, were compared. Cip6 consisted of a tandem duplication of a type I cohesin domain. Cip20 was constructed by fusing the same type I cohesin domain to the type II cohesin domain of SdbA. Cip6 was mixed with varying proportions of CelD, carrying a type I dockerin domain, and CelC-DSCelD, in which the dockerin domain of CelD was fused to the endoglucanase CelC. Likewise, Cip20 was mixed with varying proportions of CelD and CelC-DSCipA-H, which carries the type II dockerin domain of CipA. Complex formation was analyzed by non-denaturing gel electrophoresis and densitometry and the composition of the bands observed was confirmed by denaturing gel electrophoresis in a second dimension. With optimal proportions of the components, the ternary complex formed of Cip6, CelD, and CelC-DSCelD accounted for 36% of the proteins entering the gel. In contrast, up to a 56% yield was obtained for the ternary complex formed of Cip20, CelD, and CelC-DSCipA-H. The results emphasize the advantage of using different types of cohesin-dockerin pairs in the design of cellulosome-derived complexes.